Use of 4-carboxy-phthalato(1,2-diaminocyclohexane)-platinum (II) and alkali metal salts thereof with cyclophosphamide in alleviating L1210 murine leukemia

ABSTRACT

##STR1## 4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) (and alkali metal salts thereof) has shown antileukemic activity in mice against murine leukemia L1210. It is effective in dosages of 5-60 mg/kg of body weight and is potentiated in a treatment with cyclophosphamide (CY) (50 mg/kg of body weight) to which may be added 5-fluorouracil (5-FU) (75 mg/kg of body weight) or hydroxyurea (HU) (1000 mg/kg of body weight). Some previous platinum chelate compounds show dosage limitation due to renal impairment but the free carboxyl of the present Pt compound offers a possible route of oral administration and absorption by the stomach. 
     The Pt compound may be prepared by reacting the known dichloro(1,2-diaminocyclohexane)platinum(II) (NSC 194814) with silver nitrate to replace chloro with nitro. Subsequently, benzene tricarboxylic acid is added to form an off-white precipitate (2 hrs, dark, 5° C.) of the desired product which is preferably utilized as the alkali metal salt. 
     The 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) and alkali metal salts thereof may be combined in multiple drug regimen with substantially improved yield cures over the parent compound. For example, the compound denoted Pt-307 may be combined in a dual regimen with cyclophosphamide (CY) and in a triple drug regimen of Pt-307 plus cyclophosphamide (CY) and either 5-fluorouracil (5-FU) or hydroxyurea (HU) as the third component. 
     Additionally, the present compound (NSC 271674) has shown superior results in testing for renal toxicity against the parent compound (NSC 119875, cis-dichlorodiamino platinum II) and further the present compound appears to be active against strains of murine leukemia wherein the same NSC 119875 has exhausted its activity.

This is a division, of application Ser. No. 926,035, filed July 19, 1978, now pending which is a continuation-in-part of Ser. No. 828,926 filed Aug. 29, 1977, now U.S. Pat. No. 4,137,248. ##STR2##

4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) (and alkali metal salts thereof) has shown antileukemic activity in mice against murine leukemia L1210. It is effective in dosages of 5-60 mg/kg of body weight and is potentiated in a treatment with cyclophosphamide (CY) (50 mg/kg of body weight) to which may be added 5-fluorouracil (5-FU) (75 mg/kg of body weight) or hydroxyurea (HU) (1000 mg/kg of body weight). Some previous platinum chelate compounds show dosage limitation due to renal impairment but the free carboxyl of the present composition offers a possible route of oral injection and absorption by the stomach.

The compound may be prepared by reacting the known dichloro(1,2-diaminocyclohexane)platinum(II) (NSC 194814) with silver nitrate to replace chloro with nitro. Subsequently, benzene tricarboxylic acid is added to form an off-white precipitate (2 hrs, dark, 5° C.) of the desired product which is preferably utilized as the alkali metal salt.

The 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) and alkali metal salts thereof may be combined in multiple drug regimen with substantially improved yield cures over the parent compound. For example, the compound denoted Pt-307 may be combined in a dual regimen with cyclophosphamide (CY) and in a triple drug regimen of Pt-307 plus cyclophosphamide (CY) and either 5-fluorouracil (5-FU) or hydroxyurea (HU) as the third component.

It is understood in this specification and claims that where the compound 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) is utilized it is designed and meant to apply to the free acid as well as alkali metal salts thereof such as sodium, potassium, lithium, etc.

PRIOR ART

The following patents provide background material to the present invention.

U.S. Pat. No. 3,892,790 Tobe et al--The compounds disclosed are platinum(II) halogeno complexes such as cis-dichloro-Pt(II) where the remaining bonds are bis(cyclopropylamine), bit(cyclobutylamine), bis(cyclopentylamine), bis(cyclohexylamine) and bis(cycloheptylamine).

U.S. Pat. No. 3,904,663 Tobe et al--Formula 2 in the list of compounds bridging columns 1 and 2 is cyclohexane (trans)1,2-diamine platinum(II) and the compounds for claim purposes are illustrated by dichloro(ortho-phenylene-diamine)platinum(II).

The following literature references are deemed of interest:

J. M. Hill et al, "Platinum Coordination Compounds in the Treatment of Acute Leukemia and Other Malignant Diseases with Particular Reference to Malonato 1,2-Diaminocyclohexane Platinum(II)," Journal of Clinical Hematology and Oncology, 7:681-697 (1977). This compound abbreviated PHM has a structure disclosed as follows: ##STR3##

Paul Schwartz et al, "Preparation and Antitumor Evaluation of Water-Soluble Derivatives of Dichloro(1,2-Diaminocyclohexane)Platinum(II) (NSC-194814)," Cancer Treatment Reports, 61:1519-1525 (1977).

Glen R. Gale et al, "Potentiating Action of 5-Fluorouracil When Used in Combination with Platinum Compounds and Cyclophosphamide in Treatment of Advanced L1210 Leukemia," accepted for publication in Bioinorganic Chemistry.

Glen R. Gale et al, "Synergistic Action of High-Dose Hydroxyurea When Used With Cyclophosphamide and Certain New Organoplatinum Complexes in Treatment of Advanced L1210 Leukemia," accepted for publication in Cancer.

The utilization of the present compound, 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II), against L1210 murine leukemia is not anticipated by the effective prior art and it is noted that the last three literature references, one to Schwartz et al and two to Gale et al (as yet unpublished) are useful in the preparation of the present specification of invention.

The recent chemotherapy of utilizing platinum coordination compounds dates back to some work by Rosenberg and co-workers as reported in Nature, 205:698-699 (1965) and also in Nature, 222:385-386 (1969). It was early found that the cis platinous compounds had superior activity and as a starting point was mentioned cis-dichloro(1,2-diaminocyclohexane)platinum(II) known as DDCP and cis-platinous diaminodichloride (PDD). In general, newer antitumor agents modified or replaced the chlorides in dichloro-1,2-diaminocyclohexane platinum(II) (NSC 194814) by organic or inorganic anion. One difficulty with the diaminocyclohexane complex is its low solubility. It was known that the diaminocyclohexane moiety reduces the toxicity while increasing the antitumor activity of complexes with highly reactive ligands as, for example, nitrato or sulfato groups.

In the present application a dicarboxylic acid dianion (the phthalate) relates to the central platinum(II) cation and forms neutral complexes.

Additionally, while phthalato(1,2-diaminocyclohexane)platinum(II) (NSC 268255) is very insoluble, the 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) (NSC 271674) readily dissolves in 1% sodium bicarbonate solution via formation of the sodium carboxylate.

PREPARATION OF COMPOUND 4-CARBOXYPHTHALATO(1,2-DIAMINOCYCLOHEXANE)PLATINUM(II)

4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) has the following structure: ##STR4## Molecular formula: C₁₅ H₁₈ N₂ O₆ Pt Molecular weight: 517.44

This compound may be prepared by the following synthetic route: ##STR5##

A specific preparation is set out post as Example 1.

DOSAGE

Compound Pt-307 was administered usually in a dosage regimen of 5-60 mg/kg of body weight. This was given as a single dose or on Days 1, 5, and 9 in the standard National Institutes of Health 10-day testing. In this testing Pt-307 was the same compound as NSC 271674. In general, the ternary combination such as Pt+CY+FU enhanced markedly the increased lifespan of treated mice. Collectively the cure rate (greater than 60-day survival) was less than 6% with the Pt+CY combinations and was increased to over 63% upon inclusion of FU in the regimen. This particular combination was somewhat superior to that obtained with hydroxyurea (HU) below where HU was used as a third drug with the Pt+CY combination.

It is noted that 5-fluorouracil (5-FU) has also been shown to potentiate the action of irradiation. For example, mean survival times were significantly increased in patients who received combined FU and radiation treatment as compared to radiation therapy alone for adenocarcinoma of the stomach, pancreas, and large bowel. The Pt+CY+FU combinations proved to be uniquely efficaceous against advanced L1210 leukemia and specially in combination chemotherapy. CY was utilized at an optimum 50 mg/kg of body weight (range 25-75 mg/kg) preferably given at Day 3 in the 10-day testing. 5-FU was utilized at 75 mg/kg of body weight (range 50-100 mg/kg) at Day 3. HU was used in a high dosage of at least 1000 mg/kg of body weight (range 1000-1500 mg/kg). The 5-FU and the HU were utilized as ternary compositions only with cyclophosphamide and the platinum compound.

HIGH-DOSE HYDROXYUREA USED WITH CYCLOPHOSPHAMIDE AND PT NSC 271674 (Pt-307)

Hydroxyurea is utilized in combination therapy at a high dosage of at least 1000 mg/kg of body weight (range 1000-1500 mg/kg). At this dosage when used alone it is only minimally effective. In triple drug regimen involving Pt+CY and the hydroxyurea (HU), the cure rate is 11% with the dual combinations and is increased to 53% upon inclusion of HU in the regimen. It is believed that HU may inhibit a process whereby potentially lethal DNA damage produced by Pt+CY would otherwise be repaired.

EXAMPLE 1

Dichloro(1,2-diaminocyclohexane)platinum(II) (19.53 g; 0.052 mole) was suspended in 300 ml of water. Silver nitrate (15.20 g; 0.085 mole) was added and the mixture was stirred in a darkened flask overnight. The mixture was filtered and the filtrate was added to 9.90 grams of 4-carboxyphthalic acid (1,2,4-benzenetricarboxylic acid) (0.047 mole) dissolved in 200 ml of warm water. The reaction mixture was kept in a darkened flask at room temperature for two hours and then stored at 5° C. overnight. The off-white precipitate was collected, washed thoroughly with water and dried in vacuo over Drierite to yield 12.0 g (55%) of 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II).

Elemental analyses (Galbraith Laboratories, Knoxville, Tennessee):

    ______________________________________                                                   % Calculated % Found                                                 ______________________________________                                         C           34.81          34.50                                               H           3.48           3.85                                                Pt          37.70          37.08                                               ______________________________________                                    

The compound was insoluble in water but dissolved readily in 1% sodium bicarbonate solution with the evolution of carbon dioxide indicating the presence of a free carboxy group. It was also very soluble in dimethylsulfoxide (DMSO). It decomposed without melting at temperatures greater than 270° C.

Thin layer chromatography, with methanol as developer, of a DMSO solution (dissociation was suppressed by the addition of 1,2,4-benzene-tricarboxylic acid to the solution) on an Eastman 13255 cellulose chromagram sheet gave an R_(F) value of 0.75. Stannous chloride solution was the detecting agent.

The infrared spectrum of a KBr pellet gave absorbances at the following wavenumbers (cm⁻¹):

3450s, 3210s, 3100w, 2935s, 2860m, 2600w, 1720s, 1600s, 1490s, 1450m, 1350s, 1240m, 1160m, 1120m, 1090w, 1060s, 1028vw, 1025m, 980w, 915m, 860w, 840m, 800m, 760s, 700m, 670vw, 655w, 620m, 570vw, 510m, 440m, 360m, 305w.

EXAMPLE 2 Effective Dosage in Mice

The compound in 1% NaHCO₃ solution was tested against the L1210 leukemia in BDF₁ mice [10⁶ cells given to mice on Day 0, treatment (ip) on Day 1 only] in laboratory and the following % increases in life span (%ILS) were noted:

    ______________________________________                                         Dose, mg/kg  % ILS                                                             ______________________________________                                          5            70                                                               10           118                                                               20           119                                                               25           139                                                               40           232                                                               50           288 (1 of 8 mice survived                                                        > 60 days)                                                      60           174 (1 of 8 mice survived                                                        > 60 days)                                                      ______________________________________                                    

The Compound Singly and in Combination Regimen

When treatment was delayed until Day 3, 5 mg/kg gave 51% ILS, 42% ILS and 39% ILS in three experiments. In combination with cyclophosphamide (50 mg/kg) in Day 3 treatment, 5 mg/kg gave 416% ILS with 3 of 10 >60 day survivors and 217% ILS with no >60 day survivors in two experiments. In combination with cyclophosphamide (50 mg/kg) and 5-fluorouracil (75 mg/kg) in Day 3 treatment, 5 mg/kg gave 673% ILS with 8 of 10 >60 day survivors.

The approximate LD₅₀ was 75 mg/kg.

The compound was submitted to the Drug Development Branch of the National Cancer Institute and assigned NSC Number 271674. It was screened against the L1210 leukemia on a day 1, 5, 9 schedule and the following results were obtained:

    ______________________________________                                          Dose, mg/kg  % ILS      Cures (of 10)                                         ______________________________________                                         25            344        5                                                     12.5          191        2                                                     6.25          163        1                                                     3.13          46         0                                                     1.56          33         0                                                     0.78          27         0                                                     ______________________________________                                    

A second screener obtained:

    ______________________________________                                         Dose, mg/kg  % ILS                                                             ______________________________________                                         25            86                                                               12.5         147                                                               6.25          58 (2 questionably toxic deaths                                                 were noted)                                                     3.13          41                                                               1.56          19                                                               0.78          19                                                               ______________________________________                                    

No cures were obtained by the second screener.

EXAMPLE 3

A Comparison of the In Vitro Activity of NSC-119875 and NSC-271674 Against an L1210 Leukemia with Acquired Resistance to NSC-119875*

    ______________________________________                                                           ID.sub.50 (μg/ml)                                         Compound          L1210     L1210/DDP                                          ______________________________________                                         NSC-119875 (DDP)  0.05      2.50                                               NSC-271674        0.30      0.23                                               ______________________________________                                          The ID.sub.50 refers to the concentration of test compound required to         inhibit the growth of either the L1210 or L1210/DDP (NSC119875) resistant      cells by 50 percent.                                                           *Cisdichlorodiamino platinum II                                          

A Comparison of the In Vivo Activity of NSC-119875 and NSC-271674 Against a P388 Leukemia with Acquired Resistance to NSC-119875

    ______________________________________                                                        P388      P388/DDP                                                       Dose (Mg/Kg)                                                                               Survival %    Survival                                                                               %                                   Compound Days 1,5,9,13                                                                              (Days)   ILS  (Days) ILS                                  ______________________________________                                         Control  --          13.3          11.0                                        NSC-119875                                                                     (DDP)    6.7         32.6     145  15.5   41                                   NSC-271674                                                                              20.0        20.7      56  33.3   201                                  ______________________________________                                          P388/DDP refers to a tumor line with acquired resistance to NSC119875.         Data are expressed as survival in days and as a percentage increase in         life span (% ILS).                                                       

    __________________________________________________________________________      A Comparison of the Activity of NSC-119875 and                                NSC-271674 Against a Panel of a Mouse Tumors                                                 NSC-119875       NSC-271674                                                           Optimal          Optimal                                  Tumor    Schedule                                                                            Dose Range                                                                            Dose   %  Dose Range                                                                            Dose   %                                 (Site, Parameter)                                                                       (ip) (mg/kg/inj)                                                                           (mg/kg/inj)                                                                           T/C                                                                               (mg/kg/inj)                                                                           (mg/kg/inj)                                                                           T/C                               __________________________________________________________________________     B16 melanoma                                                                            D1-9 16-0.5 2      193                                                                                 25-0.39                                                                             6.25   171                               ip, ST*  D1-9 16-0.5 1      166                                                                               12.5-0.39                                                                             1.56   159                               L1210    D1-9 16-0.5 2      151                                                                                 25-0.39                                                                             0.78   174                               ip, ST   D1-9 16-0.5 2      182                                                                               12.5-0.39                                                                             1.56   325                               Lewis lung                                                                              D1-9 16-0.5 2      135                                                                                 25-0.39                                                                             3.12   122                               iv, ST   D1-9 16-0.5 2      177                                                                               12.5-0.39                                                                             6.25   127                               CD8F1 mammary                                                                           Q7DX5                                                                               16-0.5 8       0   25-0.39                                                                             6.25    11                               sc, TW** Q7DX5                                                                               32-1.0 8       0   50-1.56                                                                             25      25                               Colon #38                                                                               D2,9 16-0.5 8       42                                                                                 25-0.39                                                                             12.5    50                               sc, TW   D2,9 32-1.0 16      11                                                                                 50-1.56                                                                             50      33                               __________________________________________________________________________      *Survival time (mean or median) assay                                          **Tumor weight inhibition assay.                                               Data are expressed as a percentage of the survival time in days or the         tumor weights in mg of the treated mice divided by the corresponding           values for the control mice. All testing was performed using both platinu      compounds in the same experiment. The criteria for activity are as             follows: confirmed activity (activity from two separate laboratories) of       % T/C of 150 or more for survival systems and 10% or less for tumor weigh      inhibition assays. Lower levels of activity such as a % T/C of 125 for         survival systems and 42% for tumor weight inhibition assays are considere      statistically significant.                                               

EXAMPLE 4

    __________________________________________________________________________     Toxicity of NSC-241240, NSC-250427, NSC-256927 and                             NSC-271674 Compared to the Toxicity of NSC-119875                              in the Renal Toxicity Screening Protocol                                       Parameters Measured                                                                       NSC-119875                                                                            NSC-241240                                                                            NSC-250427                                                                            NSC-256927                                                                            NSC-271674                              __________________________________________________________________________     Body Weight Loss                                                                          3      1      2      2      3                                       Hematology-                                                                    Hematocrit 3      4      3      4      3                                       White Blood Cell                                                                          3      3      3      3      3                                       Clinical Chemistry-                                                            Blood Urea Nitrogen                                                                       3      1      1      1      1                                       Creatinine 3      1      1      1      1                                       Serum Glutamic                                                                            3      3      5      3      3                                       Pyruvic Transaminase                                                           Histopathology-                                                                Renal      3      1      2      2      2                                       Lymphatic  3      1      2      4      1                                       Hematopoietic                                                                             3      5      4      4      5                                       Gastrointestinal                                                                          3      1      1      1      1                                       TOTAL SCORE                                                                               30     21     24     25     23                                      __________________________________________________________________________      Scoring:?                                                                      1 = much less severe than NSC119875                                            2 = slightly less severe than NSC119875                                        3 = as severe as NSC119875                                                     4 = slightly more severe than NSC119875                                        5 = much more severe than NSC119875                                            NSC119875  cisdichlorodiamino platinum II                                      NSC241240  diamine [1,1cyclobutanedicarboxylato (2)0,0']platinum               NSC250427                                                                      NSC256927  bisisopropylamine-cis-dichloro-transdihydroxy                       NSC271674  4carboxyphthalato(1,2-diaminocyclohexane)-platinum(II)?       

The present compound (NSC 271674) has shown superior results in testing for renal toxicity against the parent compound (NSC 119875, cis-dichlorodiamino platinum II) and further the present compound appears to be active against strains of murine leukemia wherein the same NSC 119875 has exhausted its activity. 

We claim:
 1. A method of alleviating L1210 murine leukemia which comprises administering 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) or alkali metal salt thereof I.P. and at anti-L1210 murine leukemic dosage to a mouse of 5-60 mg/kg for up to 10 days and wherein additionally cyclophosphamide is added in an effective dosage of about 25-75 mg/kg of body weight to provide a binary treatment.
 2. The method of claim 1 wherein the additional dosage of cyclophosphamide utilized is 50 mg/kg of body weight. 